I am using a pretty old ATPase protocol that measures inorganic phosphate released by The Fiske and Subbarow method( 1925). Nothing is mentioned about ATP regenerationin the protocol. Should I add any ? and if yes, how and when do I add it? I have written the protocal below:
ATPase assay: The reaction mixture in a final volume of 0.25 ml contained 50ul of Tris buffer ( 100mM), 20ul KCl,20ul Mgcl2(125mM), 25uL EDTA( 0.4mM), ATP of 2ul and xug of protein. Incubated at 37 for - hour and reactions topped by adding 50%TCA. Then centrifuged and supernatant was estimated for pi.
0.5 ml of Sup+ 450ul of ammo.molybdate; then 0.4ml ANSA,( made in sodium bisulphate and sodium sulphate)- 15min @37. Blue colour estimated @ 660 nm..moles of inorganic phosphate formed/ ug of protein/hr.
So do I need to add anything as an ATP reg. system?
There are 2 reasons why you might need an ATP regenerating system: (1) depletion of ATP causes the reaction to slow down so that the initial rate of reaction is not maintained, or (2) accumulation of product ADP inhibits the reaction so that the initial rate is not maintained. If neither effect is observed, i.e. if the reaction progress curve is linear for the whole reaction time, then you do not need an ATP regenerating system.
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